In general, fungal plant diseases can be classified into two types: those caused by soilborne fungi and those caused by airborne fungi. Soilborne fungi cause some of the most widespread and serious plant diseases, such as root and stem rot caused by Fusarium spp. and root rot caused by Phytophthora spp. For example, Phytophthora parasitica var. nicotiana, a soilborne oomycete found in many tobacco growing regions worldwide, causes black shank, a highly destructive root and stem rot disease of many varieties of cultivated tobacco.
Since airborne fungi can be spread long distances by wind, they can cause devastating losses, particularly in crops which are grown over large regions. A number of pathogens have caused widespread epidemics in a variety of crops. Important diseases caused by airborne fungi are stem rust (Puccinia graminis) on wheat, corn smut (Ustilago maydis) on corn, and late blight disease (Phytophthora infestans) on potato and tomato. Plasmopera viticola is an airborne oomycete that causes downy mildew disease on grape vines. The blue mold fungus (Peronospora tabacina) has caused catastrophic losses in tobacco crops, particularly in the United States and Cuba.
Most of these fungal diseases are difficult to combat, and farmers and growers must use a combination of practices, such as sanitary measures, resistant cultivars, and effective fungicide against such diseases. Hundreds of millions of dollars are spent annually for chemical control of plant-pathogenic fungi. As a result, there is today a real need for new, more effective and safe means to control plant-pathogenic fungi, particularly oomycetes which are responsible for major crop loss.
Genetic engineering promises to be an effective strategy for reducing the losses associated with diseases of field crops. Several successful approaches have been reported where the constitutive expression of antimicrobial peptides such as cecropins (Arce et al., “Enhanced Resistance to Bacterial Infection by Erwinia Carotovora Susp. Atroseptica in Transgenic Potato Plants Expressing the Attacin or the Cecropin SB-37 Genes,” Am. J. Potato Res. 76:169–177 (1999)), lysozyme (Nakajima et al., “Fungal and Bacterial Disease Resistance in Transgenic Plants Expressing Human Lysozyme,” Plant Cell Reports 16:674–679 (1997)), and monoclonal antibodies (Tavladoraki et al, “Transgenic Plants Expressing a Functional Single Chain FV Antibody are Specifically Protected from Virus Attack,” Nature 366:468–472 (1993)) effectively protected plants from parasitic organisms. However successful, these approaches have limited application to food production since many of these antimicrobial peptides and plant defense molecules are potentially toxic or allergenic to humans (Franck-Oberaspach et al., “Consequences of Classical and Biotechnological Resistance Breeding for Food Toxicology and Allergenicity,” Plant Breeding 116:1–17 (1997)). Thus, alternative approaches for genetically engineering disease resistance would be more desirable.
Plants posses a highly evolved pathogen surveillance system which allows for recognition of specific pathogen derived molecules known as elicitors. Elicitor recognition results in an incompatible plant-microbe interaction, defined as the rapid activation of plant defense genes, typically resulting in the hypersensitive response and the onset of systemic acquired resistance.
The hypersensitive response is a rapid, localized necrosis that is associated with the active defense of plants against many pathogens (Kiraly, Z., “Defenses Triggered by the Invader: Hypersensitivity,” pages 201–224 in: Plant Disease: An Advanced Treatise, Vol. 5, J. G. Horsfall and E. B. Cowling, ed. Academic Press New York (1980); Klement, Z., “Hypersensitivity,” pages 149–177 in: Phytopathogenic Prokaryotes, Vol. 2, M. S. Mount and G. H. Lacy, ed. Academic Press, New York (1982)). The hypersensitive response elicited by bacteria is readily observed as a tissue collapse if high concentrations (≧107 cells/ml) of a limited host-range pathogen like Pseudomonas syringae or Erwinia amylovora are infiltrated into the leaves of nonhost plants (necrosis occurs only in isolated plant cells at lower levels of inoculum) (Klement, Z., “Rapid Detection of Pathogenicity of Phytopathogenic Pseudomonads,” Nature 199:299–300; Klement, et al., “Hypersensitive Reaction Induced by Phytopathogenic Bacteria in the Tobacco Leaf,” Phytopathology 54:474–477 (1963); Turner, et al., “The Quantitative Relation Between Plant and Bacterial Cells Involved in the Hypersensitive Reaction,” Phytopathology 64:885–890 (1974); Klement, Z., “Hypersensitivity,” pages 149–177 in Phytopathogenic Prokarvotes, Vol. 2., M. S. Mount and G. H. Lacy, ed. Academic Press, New York (1982)). The capacities to elicit the hypersensitive response in a nonhost and be pathogenic in a host appear linked. As noted by Klement, Z., “Hypersensitivity,” pages 149–177 in Phytopathogenic Prokaryotes, Vol. 2., M. S. Mount and G. H. Lacy, ed. Academic Press, New York, (1982), these pathogens also cause physiologically similar, albeit delayed, necroses in their interactions with compatible hosts. Furthermore, the ability to produce the hypersensitive response or pathogenesis is dependent on a common set of genes, denoted hrp (Lindgren, P. B., et al., “Gene Cluster of Pseudomonas syringae pv. ‘phaseolicola’ Controls Pathogenicity of Bean Plants and Hypersensitivity on Nonhost Plants,” J. Bacteriol. 168:512–22 (1986); Willis, D. K., et al., “hrp Genes of Phytopathogenic Bacteria,” Mol. Plant-Microbe Interact. 4:132–138 (1991)). Consequently, the hypersensitive response may hold clues to both the nature of plant defense and the basis for bacterial pathogenicity.
The hrp genes are widespread in Gram-negative plant pathogens, where they are clustered, conserved, and in some cases interchangeable (Willis, D. K., et al., “hrp Genes of Phytopathogenic Bacteria,” Mol. Plant-Microbe Interact. 4:132–138 (1991); Bonas, U., “hrp Genes of Phytopathogenic Bacteria,” pages 79–98 in: Current Topics in Microbiology and Immunology: Bacterial Pathogenesis of Plants and Animals—Molecular and Cellular Mechanisms, J. L. Dangl, ed. Springer-Verlag, Berlin (1994)). Several hrp genes encode components of a protein secretion pathway similar to one used by Yersinia, Shigella, and Salmonella spp. to secrete proteins essential in animal diseases (Van Gijsegem, et al., “Evolutionary Conservation of Pathogenicity Determinants Among Plant and Animal Pathogenic Bacteria,” Trends Microbiol. 1:175–180 (1993)). In E. amylovora, P. syringae, and P. solanacearum, hrp genes have been shown to control the production and secretion of glycine-rich, protein elicitors of the hypersensitive response (He, S. Y., et al. “Pseudomonas Syringae pv. Syringae HarpinPSS: a Protein that is Secreted via the Hrp Pathway and Elicits the Hypersensitive Response in Plants,” Cell 73:1255–1266 (1993); Wei, Z. -M., et al., “HrpI of Erwinia amylovora Functions in Secretion of Harpin and is a Member of a New Protein Family,” J. Bacteriol. 175:7958–7967 (1993); Arlat, M., et al. “PopA1, a Protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum,” EMBO J. 13:543–553 (1994)).
The first of these proteins was discovered in E. amylovora Ea321, a bacterium that causes fire blight of rosaceous plants, and was designated harpin (Wei, Z. -M., et al, “Harpin, Elicitor of the Hypersensitive Response Produced by the Plant Pathogen Erwinia amylovora,” Science 257:85–88 (1992)). Mutations in the encoding hrpN gene revealed that harpin is required for E. amylovora to elicit a hypersensitive response in nonhost tobacco leaves and incite disease symptoms in highly susceptible pear fruit. The P. solanacearum GMI 1000 PopA 1 protein has similar physical properties and also elicits the hypersensitive response in leaves of tobacco, which is not a host of that strain (Arlat, et al., “PopA1, a Protein Which Induces a Hypersensitive-like Response on Specific Petunia Genotypes, is Secreted via the Hrp Pathway of Pseudomonas solanacearum,” EMBO J. 13:543–53 (1994)). However, P. solanacearum popA mutants still elicit the hypersensitive response in tobacco and incite disease in tomato. Thus, the role of these glycine-rich hypersensitive response elicitors can vary widely among Gram-negative plant pathogens.
Other plant pathogenic hypersensitive response elicitors have been isolated, cloned, and sequenced. These include: Erwinia chrysanthemi (Bauer, et. al., “Erwinia chrysanthemi HarpinEch: Soft-Rot Pathogenesis,” MPMI 8(4): 484–91 (1995)); Erwinia carotovora (Cui, et. al., “The RsmA− Mutants of Erwinia carotovora subsp. carotovora Strain Ecc71 Overexpress hrpNEcc, and Elicit a Hypersensitive Reaction-like Response in Tobacco Leaves,” MPMI 9(7): 565–73 (1966)); Erwinia stewartii (Ahmad, et. al., “Harpin is not Necessary for the Pathogenicity of Erwinia stewartii on Maize,” 8th Int'l. Cong. Molec. Plant-Microb. Inter. Jul. 14–19, 1996 and Ahmad, et. al., “Harpin is not Necessary for the Pathogenicity of Erwinia stewartii on Maize,” Ann. Mtg. Am. Phytopath. Soc. Jul. 27–31, 1996); and Pseudomonas syringae pv. syringae (WO 94/26782 to Cornell Research Foundation, Inc.).
Because the hypersensitive response results in localized necrosis of plant tissue, it is desirable to limit expression of a heterologous hypersensitive response elicitor to certain tissues in transgenic plants. This approach is discussed generally in PCT publication WO 94/01546 to Beer et al., but no specific transgenic plants are identified and only two suitable fungus-responsive promoters are suggested, e.g., the phenylalanine ammonia lyase and chalcone synthase promoters. No promoters responsive specifically to infection by oomycetes are identified therein.
The present invention is directed to overcoming these and other deficiencies in the art.